CN112714794A

CN112714794A - Application of trehalase in fermentation production

Info

Publication number: CN112714794A
Application number: CN201980060900.6A
Authority: CN
Inventors: 林洁; 许宏贤; 彭辉
Original assignee: Nanjing Bestzyme Bio Engineering Co ltd
Current assignee: Nanjing Bestzyme Bio Engineering Co ltd
Priority date: 2018-09-17
Filing date: 2019-09-17
Publication date: 2021-04-27
Also published as: US20210403957A1; WO2020057476A1; US11913053B2

Abstract

Provides an application of trehalase in fermentation production, wherein the trehalase has an amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO. 8. A method for producing and using the trehalase is provided, particularly for use in the production fermentation of alcohol and amino acids.

Description

Application of trehalase in fermentation production

Technical Field

The invention relates to a process for producing a fermentation product, in particular to a polypeptide with trehalase activity and application of trehalase in fermentation production.

Background

Trehalase (α, α -trehalase, E.C 3.2.1.28) is a glycoside hydrolase that specifically hydrolyzes trehalose containing α -1,1 glycosidic linkages and releases two molecules of glucose. Trehalase is widely present in bacteria, fungi, plants and animals, and can be classified into neutral trehalase and acid trehalase according to its optimum pH, and is localized at different positions of cells, i.e., intracellularly and extracellularly. It has been shown that trehalase is present in the brush border membrane of the kidney and in the chorion of the small intestine of mammals and may be involved in the degradation of trehalose in the environment of cellular tissues. In microorganisms, trehalase also plays a crucial role in many physiological processes, such as fungal spore germination and resting cell restoration growth.

During the alcoholic fermentation process, the yeast cells combine into the protective substance trehalose under the pressure environment of high osmotic pressure and high alcoholic strength to maintain the stability of the osmotic pressure of the cells and help the cells resist the dehydration environment caused by the high osmotic pressure and high alcoholic concentration. However, trehalose accounts for about 60-70% of the disaccharides in the total residual sugars at the end of fermentation, since trehalose is not available to yeast, resulting in a large accumulation of trehalose. This part of the carbon source cannot be fermented to ethanol and becomes a limiting factor for further improvement of the ethanol yield. The addition of trehalase can convert the trehalose in the fermentation residual sugar into glucose which can be utilized by cells, and further into ethanol, and is a very effective method for reducing the residual sugar and improving the yield of alcohol. WO2016205127 reports that trehalase Ms37 can significantly increase glucose content when applied to fermentation for glucose production. Trichoderma reesei trehalase Tr65 disclosed in WO2015065978 can improve the yield of ethanol fermentation.

During the fermentation process of amino acid, a large amount of trehalose is accumulated in the strain metabolism process at the later stage of fermentation, and the fermentation production of the amino acid is very adversely affected. On one hand, a part of glucose is converted into trehalose which is difficult to utilize, so that the utilization rate of a carbon source is reduced, and on the other hand, the accumulation of a large amount of trehalose also has a plurality of adverse effects on the subsequent extraction and crystallization of amino acid. CN107058415A discloses that trehalose enzyme is added in the late stage of glutamic acid fermentation, so that the saccharic acid conversion rate of glutamic acid fermentation can be improved, the consumption of sugar is reduced, and the isoelectric crystallization can be favorably extracted after glutamic acid is fermented.

At present, few trehalases are reported to be applied to fermentation production, the fermentation production efficiency is low, and with the development of genome sequencing and biotechnology, trehalase with better properties needs to be further discovered and applied.

Disclosure of Invention

The present invention provides a method for producing a fermentation product, comprising adding a polypeptide having trehalase activity to a trehalose-containing production liquid to produce a fermentation product, the polypeptide being selected from one or more of the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of seq id no: (i) 3, 4 or 5, (ii) a cDNA sequence thereof, or (iii) a full-length complement of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the polypeptide coding sequence of SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5 or a cDNA sequence thereof.

Wherein the trehalase active polypeptide has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase active polypeptide has the amino acid sequence shown as SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO. 8. In another embodiment, the trehalase active polypeptide has the amino acid sequence shown in SEQ ID NO.6 or SEQ ID NO. 8. In one embodiment, the trehalase active polypeptide amino acid sequence is set forth in SEQ ID NO 6. In another embodiment, the trehalase active polypeptide amino acid sequence is set forth in SEQ ID NO 7. In another embodiment, the trehalase active polypeptide has the amino acid sequence shown in SEQ ID No. 8.

In some embodiments, the trehalase active polypeptide is a variant of a polypeptide as shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No.8, including substitutions, deletions and/or insertions at one or more (e.g., several) positions, or fragments of said polypeptide.

In some embodiments, the fermentation process of the invention, involving a trehalase activity polypeptide, is comprised of a polynucleotide polypeptide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of seq id no: (i) 3, 4 or 5, (ii) a cDNA sequence thereof, or (iii) a full-length complement of (i) or (ii).

In other embodiments, the fermentation process of the invention involves a trehalase active polypeptide, a polypeptide encoded by a polynucleotide that hybridizes under very stringent conditions with the mature polypeptide coding sequence of SEQ ID NO: (i) 3, 4 or 5, (ii) a cDNA sequence thereof, or (iii) a full-length complement of (i) or (ii).

In some embodiments, a trehalase active polypeptide involved in a fermentation process described herein is a polypeptide encoded by a polynucleotide having at least 65% sequence identity to the polypeptide coding sequence of SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5, or a cDNA sequence thereof. In one embodiment, the polynucleotide has at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide coding sequence of SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5, or a cDNA sequence thereof. In another embodiment, the polynucleotide sequence is the polypeptide coding sequence of SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5 or a cDNA sequence thereof.

In one embodiment, a trehalase active polypeptide is a polypeptide encoded by a polynucleotide having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a sequence encoding the polypeptide of SEQ ID No.3 or a cDNA sequence thereof. In another embodiment, the fermentation method of the present invention relates to a trehalase active polypeptide which is a polypeptide encoded by a polynucleotide which is the coding sequence of the polypeptide of SEQ ID NO.3 or the cDNA sequence thereof.

In one embodiment, a trehalase active polypeptide involved in a fermentation process described herein is a polypeptide encoded by a polynucleotide having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence encoding the polypeptide of SEQ ID NO.4 or a cDNA sequence thereof. In another embodiment, the invention relates to a trehalase active polypeptide which is a polypeptide encoded by a polynucleotide which is the sequence encoding the polypeptide of SEQ ID NO.4 or a cDNA sequence thereof.

In one embodiment, a trehalase active polypeptide is a polypeptide encoded by a polynucleotide having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a sequence encoding the polypeptide of SEQ ID No.5 or a cDNA sequence thereof. In another embodiment, the invention relates to a trehalase active polypeptide which is a polypeptide encoded by a polynucleotide which is the sequence encoding the polypeptide of SEQ ID NO.5 or a cDNA sequence thereof.

In some embodiments, the trehalase active polypeptide involved in the fermentation process of the present invention, wherein the trehalase active polypeptide having the amino acid sequence shown in SEQ ID NO.6 is derived from Thielavia terrestris; the trehalase active polypeptide with the amino acid sequence shown as SEQ ID NO.7 is derived from Myceliophthora thermophila; the trehalase active polypeptide with the amino acid sequence shown as SEQ ID NO.8 is derived from Rasamsonia emersonii.

In the fermentation method, the fermentation product is selected from alcohols and amino acids, wherein the alcohols are alcohols or ethanol, and preferably the alcohols; the amino acid is selected from glutamic acid, lysine, threonine, valine, proline, tryptophan, isoleucine or leucine, preferably glutamic acid and lysine.

In one embodiment, the fermentation product is an alcohol selected from methanol, ethanol or propanol, preferably ethanol. In one embodiment, the fermentation product is alcohol.

For ethanol fermentation production, after fermentation, the fermented slurry is distilled to extract ethanol. The ethanol obtained according to the process of the invention can be used, for example, as fuel ethanol, potable ethanol, i.e., drinkable neutral alcoholic beverages, or industrial ethanol. In another aspect, the fermentation process according to the invention produces alcohol, which includes ethanol, methanol, propanol or water.

In one embodiment, the fermentation product is an amino acid selected from the group consisting of glutamic acid, lysine, threonine, valine, proline, tryptophan, isoleucine or leucine, preferably glutamic acid and lysine. In one embodiment, the fermentation product is glutamic acid. In another embodiment, the fermentation product is lysine.

In the fermentation method of the invention, the production liquid containing trehalose is selected from saccharified liquid of alcoholic fermentation raw materials, alcoholic fermentation liquid, supernatant of alcoholic fermentation mature mash, amino acid fermentation liquid or supernatant of amino acid fermentation liquid, preferably saccharified liquid of alcoholic fermentation raw materials, supernatant of alcoholic fermentation mature mash and supernatant of amino acid fermentation.

In one embodiment, the trehalose-containing production fluid of the fermentation process is selected from the group consisting of a saccharified fluid of an alcoholic fermentation feedstock, an alcoholic fermentation broth or an alcoholic fermentation maturation mash supernatant. In another embodiment, the trehalose-containing production liquid is a saccharified liquid of an alcoholic fermentation feedstock. In another embodiment, the trehalose-containing production liquid is an alcoholic fermentation broth. In another embodiment, the trehalose-containing production liquid is an alcoholic fermentation maturation mash supernatant.

In one embodiment, the trehalose-containing production fluid of the fermentation process is selected from an amino acid fermentation broth or an amino acid fermentation supernatant. In another embodiment, the trehalose-containing production fluid is an amino acid fermentation broth. In another embodiment, the trehalose-containing production liquid is an amino acid fermentation supernatant.

In one aspect, the present invention relates to a method of producing a fermentation product, the step of producing the fermentation when the fermentation product is alcohol comprising:

(a) adding amylase to liquefy the alcohol fermentation raw material;

optionally pre-saccharifying the liquefied material prior to step (b);

(b) saccharifying the liquefied feedstock;

(c) adding yeast for fermentation;

(d) after the fermentation is finished, harvesting mature mash of alcohol;

wherein the trehalase may be present and/or added in the following steps:

a saccharification step (b);

a fermentation step (c);

simultaneous saccharification and fermentation;

after fermentation, alcohol is added into the mature mash;

optionally a pre-saccharification step prior to step (b).

In some embodiments, in the method of producing a fermentation product, the trehalase is added in an amount of 0.05 to 10U/g DS, preferably 0.1 to 5U/g DS, more preferably 0.2 to 0.5U/g DS.

In some embodiments, the trehalase is added in an amount of 0.05-10U/g DS. In some embodiments, the trehalase is added in an amount of 0.1-5U/g DS. In some embodiments, the trehalase is added in an amount of 0.2-0.5U/g DS. In some embodiments, the trehalase is added in an amount of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5U/g DS. In one embodiment, the trehalase is added in an amount of about 0.2U/g DS. In one embodiment, the trehalase is added in an amount of about 0.3U/g DS. In one embodiment, the trehalase is added in an amount of about 0.4U/g DS. In another embodiment, the trehalase is added in an amount of about 0.5U/g DS.

In some embodiments, in the method for producing a fermentation product, the fermentation step further comprises adding a saccharifying enzyme, preferably a complex saccharifying enzyme, in step (b); adding a nitrogen source in the step (c).

In one embodiment, in the method for producing a fermentation product, the amylase in the step (a) is a high temperature amylase, and the addition amount is 1-200U/g DS; the saccharifying enzyme in the step (b) is a compound saccharifying enzyme, and the adding amount is 20-600U/g DS; the yeast in the step (c) is active dry yeast, and the adding amount is 100-1500 ppm; the nitrogen source is urea, and the adding amount is 100-1000 ppm.

In one embodiment, the amylase in the step (a) is high temperature amylase, and the addition amount is 1-200U/g DS, preferably 10-100U/g DS.

In one embodiment, the saccharifying enzyme in step (b) is a complex saccharifying enzyme and is added in an amount of 20-600U/g DS, preferably 50-500U/g DS.

In one embodiment, the yeast in step (c) is active dry yeast, and is added in an amount of 100-; the nitrogen source is urea, and the adding amount is 100-1000ppm, preferably 600 ppm.

In one embodiment, in the method for producing a fermentation product, the step (a) liquefies an alcoholic fermentation raw material by adding 10 to 100U/g DS high-temperature amylase; the steps (b) and (c) are synchronously carried out, the pH acidity of the raw material liquefied liquid is adjusted, 50-500U/g DS composite saccharifying enzyme, 200-1000ppm active dry yeast, 600ppm urea and 0.2-0.5U/g DS trehalase are added, and fermentation is carried out for 48-96h at the temperature of 28-36 ℃; harvesting the alcohol matured mash.

In another embodiment, in the method for producing a fermentation product, the step (a) liquefies an alcoholic fermentation raw material by adding 10 to 100U/g DS high-temperature amylase; the steps (b) and (c) are synchronously carried out, the pH acidity of the raw material liquefaction liquid is adjusted, 50-500U/g DS composite saccharifying enzyme, 200-1000ppm active dry yeast and 600ppm urea are added, and fermentation is carried out for 48-96h at the temperature of 28-36 ℃; harvesting the mature alcohol mash, taking the supernatant, and adding 0.2-0.5U/g DS trehalase

In another aspect, the present invention provides the method for producing a fermentation product, wherein the step of producing fermentation when the fermentation product is an amino acid comprises:

(a) seed liquid culture of amino acid fermentation strains;

(b) fermenting and culturing;

(c) collecting fermentation liquor;

wherein the trehalase may be present and/or added in the following steps:

a fermentation culture step (b);

and (c) collecting fermentation liquor.

In some embodiments, the trehalase is added in an amount of 0.05-5U/ml, preferably 0.1-2.0U/ml, more preferably 0.2-1.0U/ml, most preferably 0.5U/ml. In one embodiment, the trehalase is added in an amount of 0.05-5U/ml. In one embodiment, the trehalase is added in an amount of 0.1-2U/ml.

In one embodiment, the trehalase is about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0U/ml. In one embodiment, the trehalase is added in an amount of 0.2U/ml. In one embodiment, the trehalase is added in an amount of 0.3U/ml. In one embodiment, the trehalase is added in an amount of 0.4U/ml. In one embodiment, the trehalase is added in an amount of 0.5U/ml. In one embodiment, the trehalase is added in an amount of 0.6U/ml. In one embodiment, the trehalase is added in an amount of 0.7U/ml. In one embodiment, the trehalase is added in an amount of 0.8U/ml.

In one embodiment, when the fermentation product is an amino acid, the step (a) of shake flask cultivation of the amino acid fermenting strain obtains a seed culture solution; preparing an amino acid fermentation formula in the step (b), sterilizing a fermentation culture medium, inoculating a seed culture solution, and performing fermentation culture for 24-72 hours; the step (c) obtains a fermentation broth.

In some embodiments, the trehalase is added in step (b) at the beginning of or during fermentation in an amount of 0.1-2.0U/ml, more preferably 0.2-1.0U/ml, most preferably 0.5U/ml.

In some embodiments, the trehalase is added to the supernatant of the fermentation broth obtained in step (c) in an amount of 0.1-2.0U/ml, more preferably 0.2-1.0U/ml, most preferably 0.5U/ml.

In any of the above production fermentation methods in which the fermentation product is an amino acid, the amino acid is selected from glutamic acid, lysine, threonine, valine, proline, tryptophan, isoleucine or leucine, and preferably glutamic acid and lysine.

The invention provides a method for producing a fermentation product, wherein the fermentation product is amino acid, and the production steps comprise: adding the trehalase 0.3-1U/ml into the fermentation supernatant after the amino acid fermentation, and reacting for 2-7h at pH 6.0-9.0 and temperature 32-39 deg.C.

The invention provides a method for producing a fermentation product, wherein the fermentation product is glutamic acid or lysine, and the production steps comprise: adding trehalase 0.5U/ml into the fermented supernatant after fermentation of glutamic acid or lysine, and reacting at pH 6.5-8.5 and temperature 32-37 deg.C for 5 h.

The present invention provides a method for producing fermented alcohol, the fermentation step comprising:

(a) adding amylase to liquefy the alcohol fermentation raw material;

optionally pre-saccharifying the liquefied material prior to step (b);

(b) saccharifying the liquefied feedstock;

(c) adding yeast for fermentation;

(d) after the fermentation is finished, harvesting the mature alcohol mash;

wherein the method comprises the presence and/or addition of trehalase in the following steps:

a saccharification step (b);

a fermentation step (c);

simultaneous saccharification and fermentation;

harvesting mature alcohol mash after fermentation;

optionally a pre-saccharification step prior to step (b);

the trehalase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.

In some embodiments, the above method of producing fermented alcohol, the trehalase has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase has at least 90% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In another embodiment, the trehalase has at least 91% sequence identity to the amino acid sequence shown in SEQ ID NO 6, SEQ ID NO.7 or SEQ ID NO. 8. In one embodiment, the trehalase has at least 92% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase has at least 93% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase has at least 94% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase has at least 95% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase has at least 96% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase has at least 97% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase has at least 98% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8. In one embodiment, the trehalase has at least 99% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.

In the above method for producing fermented alcohol, the trehalase has an amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO. 8.

In some embodiments, in the above method for producing fermented alcohol, the step (b) comprises adding a saccharifying enzyme, preferably a complex saccharifying enzyme; said step (c) comprises adding nitrogen.

In one embodiment, in the above method for producing fermented alcohol, the amylase in step (a) is a high temperature amylase, and the addition amount is 1-200U/g DS; the saccharifying enzyme in the step (b) is a compound saccharifying enzyme, and the adding amount is 20-600U/g DS; the yeast in the step (c) is active dry yeast, and the adding amount is 100-1500 ppm; the nitrogen source is urea, and the adding amount is 100-1000 ppm.

In one embodiment, in the above method for producing fermented alcohol, 10 to 100U/g DS high temperature amylase is added to liquefy the alcohol fermentation raw material in step (a); the steps (b) and (c) are synchronously carried out, the pH acidity of the raw material liquefied liquid is adjusted, 50-500U/g DS composite saccharifying enzyme, 200-1000ppm active dry yeast, 600ppm urea and 0.2-0.5U/g DS trehalase are added, and fermentation is carried out for 48-96h at the temperature of 28-36 ℃; harvesting the alcohol matured mash.

In one embodiment, in the above method for producing fermented alcohol, 10 to 100U/g DS high temperature amylase is added to liquefy the alcohol fermentation raw material in step (a); the steps (b) and (c) are synchronously carried out, the pH acidity of the raw material liquefaction liquid is adjusted, 50-500U/g DS composite saccharifying enzyme, 200-1000ppm active dry yeast and 600ppm urea are added, and fermentation is carried out for 48-96h at the temperature of 28-36 ℃; and (d) after the alcohol mature mash is harvested, adding 0.2-0.5U/g DS trehalase.

The preparation method of the trehalase comprises the following steps:

a DNA construct comprising a nucleic acid encoding an enzyme can be configured for expression in a host cell. Because of the well-known degeneracy in genetic coding, different polynucleotides encoding the same amino acid sequence can be designed and prepared using routine skill. Optimization of codons for a particular host cell is also well known in the art. The nucleic acid encoding the enzyme may be incorporated into a vector.

Construction of trehalase expression plasmid: selecting plasmid vectors, exemplary plasmids being pUC19, pUC 57; the nucleic acid encoding the enzyme can be operably linked to a suitable promoter to allow transcription in a host cell, and the expression vector can further comprise a suitable transcription terminator; the vector may also comprise a selectable marker, e.g. a gene whose product complements a defect in the isolated host cell, and the vector may comprise an Aspergillus selectable marker such as amdS, argB. The vector may also comprise a DNA sequence which allows the vector to replicate in the host cell, an exemplary sequence of such a sequence being the origin of replication of plasmids pUC19, pUC57, or pUB 110.

In one embodiment, the construction of the trehalase expression plasmid comprises the following parts:

(1) carrying out PCR on the pUC57 plasmid through vector-F and vector-R primers to obtain a linearized vector fragment;

(2) a selection marker amdS expression cassette;

(3) a DNA fragment containing a gla promoter and a terminator of an Aspergillus niger glucoamylase gene;

(4) the trehalase gene is respectively derived from 3 fungi, wherein the trehalase gene derived from Thielavia terrestris is subjected to codon optimization to obtain a sequence of Thi37 (the nucleotide sequence is SEQ ID NO.3, and the amino acid sequence is SEQ ID NO.6), the trehalase gene derived from Myceliopthora thermophila is subjected to codon optimization to obtain a sequence of Myc37 (the nucleotide sequence is SEQ ID NO.4, and the amino acid sequence is SEQ ID NO.7), and the trehalase gene derived from Rasamsonia emersonii is subjected to codon optimization to obtain a sequence of Tem65 (the nucleotide sequence is SEQ ID NO.5, and the amino acid sequence is SEQ ID NO. 8).

First, amdS gene with recombination arms and DNA fragment containing gla promoter and terminator were amplified by PCR using primers amdS-F and amdS-R, gla-F and gla-R, respectively, and the linearized pUC57 vector, amdS gene and gla promoter and terminator DNA fragments were recombined by Gibson Master Mix Kit (E2611, New England Biolabs) to obtain pGla-amdS plasmid. The plasmid can be used for the insertion of trehalase gene after being linearized by AflII site.

The trehalase expression vector Thi37 was constructed as follows: the Thi37 gene with a recombinant arm was PCR-amplified with primers Thi37-F and Thi37-R, and then the Thi37 gene was recombined with the linearized pGla-amdS plasmid by means of the Gibson Master Mix Kit (E2611, New England Biolabs) to give the pThi37-amdS plasmid.

The trehalase expression vector Myc37 was constructed as follows: the Myc37 gene with a recombinant arm was amplified by PCR using primers Myc37-F and Myc37-R, and then recombined with linearized pGla-amdS plasmid using Gibson Master Mix Kit (E2611, New England Biolabs) to give pMyc37-amdS plasmid.

Trehalase expression vector Tem65 was constructed as follows: the primer Tem65-F and Tem65-R were used to PCR amplify the Tem65 gene with recombination arms, and then the Tem65 gene and the linearized pGla-amdS plasmid were recombined by Gibson Master Mix Kit (E2611, New England Biolabs) to obtain pTem65-amdS plasmid.

Transformation and integration of trehalase expression cassette: the method is characterized in that three trehalase expression cassettes are respectively introduced into an Aspergillus niger CICC2462 strain by a protoplast transformation method, and comprises the following steps: (1) preparation of protoplasts as is conventional in the art; (2) and (3) protoplast transformation, mixed transformation of the DNA fragment containing the trehalase expression cassette obtained by ApaI linearization, and selection of positive transformants in acetamide culture medium.

3 trehalase-positive transformants were obtained by transforming 3 trehalase expression cassettes, Thi37-amdS, Myc37-amdS and Tem65-amdS, respectively, into the A.niger strain.

Trehalase expression: trehalase Aspergillus niger recombinant expression strains are cultured by shake flask fermentation to obtain trehalase fermentation liquor. Trehalase can be obtained by conventional purification methods.

Interpretation of terms

Trehalase active polypeptide or trehalase refers to a polypeptide capable of specifically hydrolyzing trehalose containing alpha-1, 1 glycosidic linkages and releasing two molecules of glucose. In the present invention, the trehalase is derived from Thielavia terrestris trehalase, Myceliophthora thermophila trehalase, and Rasamsonia emersonii trehalase. In one embodiment, the trehalase active polypeptide is a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No.6, and is derived from Thielavia terrestris, having trehalase activity. In one embodiment, the trehalase active polypeptide is a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No.7, and is derived from Myceliophthora thermophila, having trehalase activity. In one embodiment, the trehalase active polypeptide is a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No.8, and the polypeptide is derived from Rasamsonia emersonii having trehalase activity.

The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein", and "peptide", and is used interchangeably. In the case where such amino acid sequences exhibit activity, they are referred to as "enzymes". The conventional single letter code or three letter code for amino acid residues is used, wherein the amino acid sequence is presented in the standard amino to carboxyl terminal orientation (i.e., N → C).

The term "sequence identity" means that the relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". When aligned using the CLUSTALW algorithm at preset parameters, a particular sequence has at least a certain percentage of amino acid residues that are identical to the amino acid residues of a given reference sequence. See Thompson et al (1994) nucleic acids as cidsRes.22: 4673-4-4680. The CLUSTALW algorithm has the preset parameters as follows: deletion counts are residues that are not identical compared to the reference sequence. Including deletions occurring at any terminus. For example, a 500 amino acid residue polypeptide that lacks the five amino acid residues at the C-terminus has a percentage sequence identity of 99% (495/500 identical residues x 100) relative to the parent polypeptide. Such variants are encompassed by the language "having at least 99% sequence identity".

The term "high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65 ℃.

The term "very high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70 ℃.

The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. The cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps including splicing and then appears as mature spliced mRNA.

The term "alcoholic fermentation feedstock" refers to a starting material selected based on the desired fermentation product (alcohol, i.e., ethanol). Examples of starch-containing starting materials suitable for use in the process of the present invention include cereals, tubers or grains. Specifically, the starch-containing material may be corn, wheat, barley, rye, sorghum, sago, tapioca (cassava), tapioca (tapioca), sorghum, oat, rice, pea, bean, or sweet potato, or a mixture thereof. Waxy and non-waxy types (waxy and non-waxy types) of corn and barley are also contemplated. In one embodiment, the alcoholic fermentation feedstock is corn. In another embodiment, the alcoholic fermentation feedstock is wheat.

The term "liquefaction liquid" refers to a starch feedstock that has been subjected to a liquefaction process. The term "saccharified liquid of alcoholic fermentation raw material" refers to a slurry obtained by saccharifying a liquefied alcoholic fermentation raw material. The term "slurry" refers to an aqueous mixture containing insoluble solids.

The term "alcoholic fermentation broth" refers to an aqueous slurry of fermentation feedstock in the presence of microbial organisms, such as ethanologenic microorganisms, and at least one enzyme, such as amylase, in the production of alcohol.

The term "alcohol matured mash" refers to raw materials in alcoholic fermentation that are fermented by adding ethanol microorganisms, and the fermented mash after fermentation is finished is the alcohol matured mash.

The term "supernatant of alcohol fermentation mature mash" refers to supernatant obtained by adding ethanol microorganism and the like to raw materials in alcohol fermentation for fermentation, wherein the fermented mash after fermentation is the alcohol mature mash, and the alcohol mature mash is subjected to standing, centrifuging and the like.

The term "amylase" or "amylolytic enzyme" refers to an enzyme that is capable of catalyzing, inter alia, the degradation of starch. Including but not limited to alpha-amylases, beta-amylases, alpha-glucosidases (EC 3.2.1.20; alpha-D-glucosinolates glucohydrolases), glucoamylases (EC 3.2.1.3; alpha-D- (1 → 4) -glucan glucohydrolases) and specific product amylases such as maltotetraglucosidases (EC 3.2.1.60) and maltohexaosidases. High temperature amylases refer to amylases which remain active when exposed to higher temperatures, and are generally thermostable or thermostable.

The term "amino acid fermenting strain" refers to a fermentation-producing strain commonly used in the fermentative production of amino acids, and generally includes Bacillus strains. For example, fermentation strains of glutamic acid include, but are not limited to, Corynebacterium glutamicum, Brevibacterium tianjingensis, Corynebacterium crenatum, Corynebacterium pekinense and mutants thereof; lysine fermentation strains include, but are not limited to, strains such as Corynebacterium glutamicum, Brevibacterium flavum, Corynebacterium crenatum, Escherichia coli; threonine fermenting strains include, but are not limited to, corynebacterium glutamicum, brevibacterium lactofermentum (b.lactofermentum), escherichia coli; proline fermenting strains include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum; valine fermenting strains including but not limited to yellow Bacillus brevis, glutamic acid Corynebacterium, lactic acid fermentation Bacillus brevis; tryptophan includes, but is not limited to, escherichia coli, corynebacterium glutamicum; isoleucine includes, but is not limited to, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium glutamicum; and isoleucine including but not limited to Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium glutamicum.

The term "amino acid fermentation liquid" refers to amino acid fermentation liquid obtained by inoculating an amino acid fermentation strain into an amino acid fermentation medium to perform fermentation culture, and producing and accumulating a specific amino acid, wherein the fermentation liquid containing the medium, the thallus and a fermentation product in the process is the amino acid fermentation liquid.

The term "supernatant of amino acid fermentation broth" refers to the supernatant obtained by centrifugation or membrane treatment of the fermentation broth after the amino acid fermentation to remove bacteria and insoluble substances.

The term "complex saccharifying enzyme": the compound enzyme is a combination of more than two enzymes, one enzyme takes a raw material as a substrate, the other enzyme takes a product of the first enzyme as a substrate, and the enzymes catalyze a series of reactions together to finally obtain a required product. The composite saccharifying enzyme is an enzyme preparation formed by mixing amyloglucosidase and pullulanase according to a certain proportion, wherein the pullulanase acts on debranching, and the amyloglucosidase hydrolyzes and liquefies starch to obtain glucose. The pullulanase composite saccharifying enzyme is prepared by compounding high-efficiency composite saccharifying enzyme and pullulanase with wide pH adaptability and good thermal stability according to a proper proportion. Wherein the pullulanase is prepared by fermenting Bacillus subtilis and can quickly hydrolyze alpha-D-1, 6 glucosidic bonds in starch to generate straight-chain dextrin; the saccharifying enzyme is prepared by fermenting Aspergillus niger (Aspergillus niger), can rapidly hydrolyze alpha-D-1, 4 glycosidic bond in liquefied starch, and can also slowly hydrolyze alpha-D-1, 6 glycosidic bond to generate glucose. For example, the PersongHighDEX ultraor PersongHighDEX SP efficient complex glucoamylase.

The term "specific activity" refers to the number of moles of substrate that can be converted to a product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed in units (U)/mg protein.

The term "dry solids content (DS)" refers to the total solids of the slurry in dry weight percent.

The phrase "Simultaneous Saccharification and Fermentation (SSF)" refers to a process of producing a biochemical in which a microbial organism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present during the same processing step. SSF involves the contemporaneous hydrolysis of a starch substrate (granular, liquefied, or solubilized) into sugars, including glucose, and fermentation of the sugars into alcohol or other biochemicals or biomaterials in the same reactor vessel.

The term "about" refers to ± 10% of the referenced value.

Drawings

FIG. 1A pThi37-amds plasmid map;

FIG. 1B a plasmid map of pMyc 37-amds;

FIG. 1C pTem65-amds plasmid map;

FIG. 2A stability of trehalase at 32 ℃;

FIG. 2B stability of trehalase at 37 ℃;

FIG. 2C stability of trehalase at 60 ℃;

FIG. 3 stability of trehalase at pH 4.0.

Detailed Description

Example 1.3 construction of trehalase expression plasmids, all of which comprise the following parts:

(1) linearizing the pUC57 plasmid through vector-F and vector-R primers;

(2) the selection marker amdS expression cassette is synthesized by GenScript company, and the sequence is shown in SEQ ID NO. 1;

(3) the DNA fragment containing the gla promoter and terminator of the Aspergillus niger glucoamylase gene is synthesized by GenScript company, and the sequence is shown in SEQ ID NO. 2;

First, amdS gene with recombination arms and DNA fragment containing gla promoter and terminator were amplified by PCR using primers amdS-F, amdS-R, gla-F and gla-R, respectively, and the linearized pUC57 vector, amdS gene and gla promoter and terminator DNA fragments were recombined by Gibson Master Mix Kit (E2611, New England Biolabs) to obtain pGla-amdS plasmid, which was sequenced to confirm the correct sequence. The plasmid can be used for the insertion of trehalase gene after being linearized by AflII site.

The trehalase expression vector Thi37 was constructed as follows: the primer Thi37-F and Thi37-R are used for PCR amplification of the Thi37 gene with a recombination arm, the Thi37 gene and the linearized pGla-amdS plasmid are recombined through a Gibson Master Mix Kit (E2611, New England Biolabs) to obtain the pThi37-amdS plasmid, the sequence is confirmed through sequencing, and the constructed plasmid map is shown in figure 1A. The plasmid can be linearized by an ApaI site and used for protoplast transformation.

The trehalase expression vector Myc37 was constructed as follows: myc37 gene with recombination arm is amplified by PCR with primers Myc37-F and Myc37-R, and then the Myc37 gene and linearized pGla-amdS plasmid are recombined by Gibson Master Mix Kit (E2611, New England Biolabs) to obtain pMyc37-amdS plasmid, the sequence is confirmed by sequencing, and the constructed plasmid map is shown in figure 1B. The plasmid can be linearized by an ApaI site and used for protoplast transformation.

Trehalase expression vector Tem65 was constructed as follows: the primer Tem65-F and Tem65-R are used for PCR amplification of a Tem65 gene with a recombination arm, then the Tem65 gene and a linearized pGla-amdS plasmid are recombined through a Gibson Master Mix Kit (E2611, New England Biolabs) to obtain pTem65-amdS plasmid, the sequence is confirmed through sequencing, and the constructed plasmid map is shown in figure 1C. The plasmid can be linearized by an ApaI site and used for protoplast transformation.

The sequences of the relevant primers are as follows:

TABLE 1 primers in this patent

Example 2 transformation integration of trehalase expression cassettes

3 trehalase expression cassettes are respectively introduced into an Aspergillus niger CICC2462 strain (purchased from China center for culture Collection of Industrial microorganisms) by a protoplast transformation method, and the specific operation steps are as follows:

(1) preparation of protoplast: inoculating Aspergillus niger mycelia in a nutrient-rich TZ liquid culture medium (beef extract powder 0.8%, yeast extract 0.2%, peptone 0.5%, NaCl 0.2%, sucrose 3%, pH 5.8), culturing for 48h, filtering with Mira-broth (Calbiochem Co., Ltd.) to collect mycelia, and washing with 0.7M NaCl (pH 5.8); after the mycelium is filtered to be dry, transferring the mycelium into enzymolysis liquid (pH 5.8) containing 1 percent of cellulase (Sigma), 1 percent of helicase (Sigma) and 0.2 percent of lywallzyme (Sigma), and carrying out enzymolysis for 3h at 30 ℃ and 65 rpm; then the enzymatic hydrolysate containing protoplast was placed on ice and filtered with four layers of mirror paper, the resulting filtrate was centrifuged gently at 3000rpm and 4 ℃ for 10min, the supernatant was discarded, and the protoplast adhered to the tube wall was treated with STC solution (1M D-Sorbitol, 50mM CaCl)₂10mM Tris, pH 7.5) and finally resuspending the protoplasts in the appropriate amount of STC solution.

(2) Protoplast transformation: 10 μ l (concentration: 1000 ng/mul) DNA fragment containing trehalase expression cassette obtained by ApaI linearization is added into 100 mul protoplast suspension liquid, mixed uniformly and placed for 25min at room temperature, then 900 mul PEG solution is added in 3 times, mixed uniformly and placed for 25min at room temperature, then centrifuged for 10min at room temperature and 3000rpm, supernatant is discarded, the protoplast attached to the tube wall is resuspended in 1ml STC solution, and then mixed with acetamide culture medium (sucrose 3%, KCl 0.05%, K) cooled to about 45 ℃ in advance₂HPO ₄·3H ₂O 0.1％、FeSO ₄ 0.001％、MgSO ₄0.0244%, acetamide 0.06% and CsCl 0.34%), laying flat, placing in 34 deg.C incubator for 4-5 days after the flat is solidified, selecting transformant to new acetamide culture medium flat, placing in 34 deg.C incubator for 4-5 days, and obtaining transformant as positive transformant.

By adopting the protoplast transformation method, 3 trehalase expression cassettes Thi37-amdS, Myc37-amdS and Tem65-amdS are respectively transformed into an Aspergillus niger strain to obtain 3 trehalase positive transformants.

Example 3 Shake flask culture of trehalase Aspergillus niger recombinant expression strains

The 3 positive transformants were inoculated into 50ml YPM medium (yeast extract 0.2%, peptone 0.2%, maltose 2%) shake flasks, shake-cultured at 34 ℃ and 220rpm for 6 days, and the supernatant of the fermentation broth was collected by centrifugation and subjected to trehalase enzyme activity assay.

Example 4 determination of enzyme Activity of trehalase

In the enzyme reaction system, trehalase can hydrolyze 1 molecule of trehalose into 2 molecules of glucose, and the produced glucose is a reducing sugar and can be measured by the DNS color method. Trehalase enzyme activity definition: the amount of enzyme required to produce 1umol of glucose per minute at a pH of 4.0 and a temperature of 37 ℃ is one unit of enzyme activity.

The enzyme activity determination method comprises the following steps: diluting the enzyme solution with appropriate amount of acetic acid-sodium acetate buffer (pH4.0, 0.05M), placing 1.0ml in a test tube, adding 1.0ml of 1% trehalose dissolved with acetic acid-sodium acetate buffer (pH4.0, 0.05M), immediately placing the test tube in water bath at 37 deg.C for heat preservation, accurately reacting for 30min, immediately taking out, adding 2.5ml DNS color developing solution (Miller 1959), boiling for 10min, cooling, adding 8ml distilled water, mixing, and measuring the absorbance of the sample at 540nm wavelength with spectrophotometer.

Through shake flask activity screening, a trehalase THI37 high-expression strain ANTHI37, a trehalase MYC37 high-expression strain ANMYC37 and a trehalase TEM65 high-expression strain ANTEM65 are obtained.

Taking the trehalase THI37 high expression strain ANTHI37 to carry out protein electrophoresis (SDS-PAGE) on supernatant of shake flask culture fermentation broth, and observing that the molecular weight of the trehalase THI37 is about 85kDa, and the enzyme activity of the trehalase in the supernatant of the fermentation broth is 1176U/ml.

And (3) carrying out protein electrophoresis (SDS-PAGE) on the supernatant of the shake-flask culture fermentation broth of the trehalase MYC37 high-expression strain ANMYC37, and observing that the molecular weight of the trehalase MYC37 is about 90kDa and the enzyme activity of the trehalase in the supernatant of the fermentation broth is 682U/ml.

Taking the trehalase TEM65 high expression strain ANTEM65 to carry out protein electrophoresis (SDS-PAGE) on the supernatant of shake flask culture fermentation broth, and observing that the molecular weight of the trehalase TEM65 is about 120kDa, and the enzyme activity of the trehalase in the supernatant of the fermentation broth is 1488U/ml.

Example 5 analysis of the enzymatic Properties of trehalase:

(1) protein concentration was determined by Coomassie Brilliant blue method (Bradford 1976).

The specific activity of trehalase THI37 is 184.03U/mg, the specific activity of trehalase MYC37 is 166.73U/mg, and the specific activity of trehalase TEM65 is 310.13U/mg.

Trehalase gene Ms37 is derived from Myceliophthora sepedonium and has the sequence of SEQ ID NO.9, see patent WO 2016205127; the trehalase gene Tr65 is derived from Trichoderma reesei, has a sequence of SEQ ID NO.10, and is referred to patent WO 2013148993. Trehalase Ms37 and Tr65 expressed in A.niger according to the methods of examples 1 and 2 were used as controls in comparison to the 3 trehalases in the present method. The specific activity of trehalase Ms37 was 207.23U/mg, and the specific activity of trehalase Tr65 was 361.06U/mg.

(2) Trehalase optimum temperature analysis

The enzyme activity of the different trehalase solutions is respectively measured at 25 ℃, 30 ℃, 37 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, the method adopts the trehalase enzyme activity measuring method, each sample is provided with three repetitions, and the highest point of the enzyme activity is the optimal reaction temperature of the enzyme.

As shown in Table 2, the optimum reaction temperature for trehalase THI37 was 50 ℃, the optimum reaction temperature for trehalase MYC37 was 60 ℃ and the optimum reaction temperature for trehalase TEM65 was 60 ℃. Compared with trehalase Ms37 and Tr65, trehalase THI37, MYC37 and TEM65 have wider temperature adaptation range and better temperature suitability.

TABLE 2 optimum temperature analysis of different trehalases

(3) Trehalase temperature stability assay

And (3) respectively preserving the different trehalase solutions at the temperature of 32 ℃, 37 ℃ and 60 ℃ for 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 16 hours, 24 hours, 30 hours, 48 hours, 54 hours and 72 hours, and then determining the enzyme activity according to the trehalase activity measuring method, wherein each sample is provided with three repetitions, and a thermal stability curve of the enzyme is drawn by taking the non-preserved enzyme solution as a reference.

The results are shown in FIG. 2: the relative enzyme activities of trehalase THI37, MYC37 and TEM65 are higher than that of trehalase Ms37 within the same time of heat preservation at 32 ℃, 37 ℃ and 60 ℃, which shows that trehalase THI37, MYC37 and TEM65 are more stable than that of Ms37 under different temperature conditions.

(4) Determination of optimum pH of trehalase

Preparing buffer solutions with different pH values (the pH values are respectively 2.5, 3.0, 3.5, 4, 4.5, 5.0, 5.5, 6, 6.5, 7, 7.5 and 8), diluting the trehalase enzyme solution to proper concentrations by using the buffer solutions with different pH values to obtain trehalase dilution solutions with different pH values, measuring the enzyme activity in the buffer solutions with different pH values according to the trehalase activity measuring method, and drawing a relative enzyme activity curve.

The results are shown in Table 3: the optimum reaction pH of trehalase THI37 was 4.5, the optimum reaction pH of trehalase MYC37 was 4.0, and the optimum reaction pH of trehalase TEM65 was 5.0. Compared with trehalase Ms37, trehalase THI37, MYC37 and TEM65 have wider pH adaptation range and better pH adaptation.

TABLE 3 optimum pH analysis of different trehalases

(5) Determination of pH stability

Diluting the trehalase solution to a proper concentration by using a buffer solution with pH4.0, keeping the temperature at 32 ℃ for 2 hours, 6 hours, 24 hours, 48 hours, 54 hours and 72 hours, and determining the enzyme activity according to the trehalase activity measuring method, wherein three times are set for each sample, and a pH stability curve is drawn.

The results are shown in FIG. 3: the relative enzyme activities of trehalase THI37, MYC37 and TEM65 are all higher than that of trehalase Ms37 when the temperature is kept for the same time under the condition of pH4.0, which indicates that the trehalase THI37, MYC37 and TEM65 are more stable than that of Ms37 under the condition.

Example 6 addition of trehalase to fermentation supernatant after completion of alcohol fermentation

The mature mash of alcohol from an alcohol production plant is centrifuged, the supernatant is taken, and the trehalose content in the supernatant is determined by ion chromatography to be 2551 mg/L. And (3) taking a proper amount of the supernatant, adjusting the pH to 4.0, and subpackaging the supernatant into 5ml centrifuge tubes, wherein the content of the supernatant in each centrifuge tube is 3 ml. Trehalase Tr65 expressed in Aspergillus niger according to the methods of examples 1 and 2 was used as a control, and 3 trehalases of the present invention were added to the centrifuge tube in an amount of 0.2U/g DS. The control group did not add trehalase. Reaction conditions are as follows: 32 ℃ and 18 h. After the reaction is finished, carrying out enzyme inactivation in boiling water bath for 10min, and then detecting the content of trehalose by ion chromatography. The results of the experiment are shown in table 4: the trehalose enzyme THI37 has the best effect of hydrolyzing trehalose in alcohol mature mash, is obviously superior to the trehalose enzyme Tr65, and can hydrolyze 100 percent of trehalose in fermentation liquor when the reaction is finished; the trehalose enzymes MYC37 and TEM65 have significantly better effects on hydrolyzing trehalose in alcohol mature mash than the trehalose enzyme Tr65, and can respectively hydrolyze 91.7% and 92.6% of trehalose in fermentation liquor after the reaction is finished.

TABLE 4 ion chromatography analysis results of alcohol matured mash

Example 7 Effect of addition of trehalase on alcohol fermentation of corn starch

Liquefying an alcoholic fermentation raw material: taking a certain amount of whole-ground corn flour (purchased from a certain alcohol factory) to prepare a mixture with a water ratio of 1: 2.3 of the feed liquid. After the preparation is finished, the pH value is adjusted to be 5.6, and a proper amount of high-temperature amylase (monolithol X5) is added, wherein the addition amount is 10-100U/g DS for liquefaction. Liquefaction conditions: the temperature is 95 ℃ and the time is 120 min.

Alcohol fermentation: the liquefied feed liquid is cooled to room temperature in time, the pH value is adjusted to 4.3 (the pH value is adjusted by 1mol/L hydrochloric acid or 3mol/L sodium hydroxide solution), the feed liquid is uniformly subpackaged into a shake flask, and 50-500U/g DS composite saccharifying enzyme (Baishijie high DEX ultra), 200-1000ppm active dry yeast (brewing high activity dry yeast, purchased from Angel Yeast Co., Ltd.) and 600pmm nitrogen source urea are added for carrying out corn alcohol fermentation. The trehalase was added to the experimental group at the beginning of the fermentation at an amount of 0.5U/g DS, while the trehalase was not added to the control group. The fermentation condition is 32 ℃ and the time is 72 h. After fermentation, detecting the content of each component such as ethanol in the fermentation liquor by high performance liquid chromatography, and taking part of mash to measure residual total sugar. The results of the experiment are shown in table 5: the addition of trehalase in the fermentation process is beneficial to improving the alcohol yield, wherein the trehalase THI37 has the best effect, the alcohol yield is improved by 1.43% compared with a control group without trehalase, and the concentration of residual total sugar is obviously reduced after the fermentation is finished; the effect of adding trehalase TEM65 was comparable to trehalase Tr65, the alcohol yield was increased by 1.29% compared to the control without trehalase, and the residual total sugar concentration was significantly reduced at the end of the fermentation.

Trehalase is added during the pre-saccharification process (start, middle, end) of the fermentation, and both the increase in alcohol yield and the decrease in the residual total sugar concentration at the end of the fermentation are achieved during the yeast fermentation (start, middle, end) and during the fermentation while saccharifying.

TABLE 5 high performance liquid chromatography analysis results of alcoholic fermentation liquors

Example 8 addition of trehalase to fermentation supernatant from glutamic acid fermentation

The glutamic acid fermentation liquor from a certain factory is centrifuged to obtain supernatant, and the trehalose content in the supernatant is determined to be 4504mg/L by ion chromatography. And (3) taking a proper amount of the supernatant and subpackaging the supernatant into 5ml centrifuge tubes, wherein the content of the supernatant in each centrifuge tube is 3 ml. 4 different trehalases were added to the centrifuge tubes in an amount of 0.5U/ml supernatant. Reaction conditions are as follows: the reaction time is 5h at the pH of 6.8 and the temperature of 37 ℃. And detecting the content of trehalose by ion chromatography after the reaction is finished. The results of the experiment are shown in table 6: the trehalose is best hydrolyzed in glutamic acid fermentation liquor by the trehalose enzyme THI37, the effect is obviously better than that of the trehalose enzyme Tr65, and the trehalose enzyme THI37 can hydrolyze 91.0 percent of the trehalose in the fermentation liquor after the reaction is finished. The addition of trehalase in the glutamic acid fermentation process can also help to degrade trehalose in the fermentation liquor and improve the utilization rate of sugar.

TABLE 6 ion chromatography analysis results of glutamic acid fermentation broth

Example 9 addition of trehalase to fermentation supernatant from completion of lysine fermentation

The lysine fermentation liquor from a certain factory is centrifuged to obtain the supernatant, and the trehalose content in the supernatant is measured by ion chromatography to be 5427 mg/L. And (3) taking a proper amount of the supernatant and subpackaging the supernatant into 5ml centrifuge tubes, wherein the content of the supernatant in each centrifuge tube is 3 ml. 5 different trehalases were added to the centrifuge tubes in an amount of 0.5U/ml supernatant. Reaction conditions are as follows: the reaction time is 5h at 37 ℃ and pH 7.39. And detecting the content of trehalose by ion chromatography after the reaction is finished. The results of the experiment are shown in table 7: the trehalose enzyme MYC37 has the best effect of hydrolyzing trehalose in lysine fermentation liquor, is obviously superior to the trehalose enzyme Tr65, and can hydrolyze 88.1 percent of trehalose in the fermentation liquor when the reaction is finished; the trehalose enzyme THI37 has a significantly better trehalose hydrolyzing effect than the trehalose enzyme Tr65 in glutamic acid fermentation broth. The addition of trehalase in the lysine fermentation process can also help to degrade trehalose in the fermentation liquor and improve the utilization rate of sugar.

TABLE 7 ion chromatography results of lysine fermentation

Claims

A method of producing a fermentation product, the method comprising adding a polypeptide having trehalase activity to a production liquor comprising trehalose to produce a fermentation product, the polypeptide being selected from one or more of the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8;

(b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of seq id no: (i) 3, 4 or 5, (ii) a cDNA sequence thereof, or (iii) a full-length complement of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the polypeptide coding sequence of SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5 or a cDNA sequence thereof.
The method of claim 1, wherein the trehalase active polypeptide has at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.
The method of claim 2, wherein the amino acid sequence of the trehalase-active polypeptide is shown in SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO. 8.
The method of claim 1, wherein said trehalase active polypeptide is a polypeptide encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) the mature polypeptide coding sequence of seq id no: (i) 3, 4 or 5, (ii) a cDNA sequence thereof, or (iii) a full-length complement of (i) or (ii).
The method of claim 1, wherein the trehalase active polypeptide is a polypeptide encoded by a polynucleotide having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the polypeptide coding sequence of SEQ ID NO 3 or SEQ ID NO 4 or SEQ ID NO 5 or a cDNA sequence thereof.
The method of claim 5, wherein the polynucleotide sequence is the polypeptide coding sequence of SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5 or a cDNA sequence thereof.
The method according to claim 3, wherein the trehalase active polypeptide having the amino acid sequence as shown in SEQ ID NO 6 is derived from Thielavia terrestris; the trehalase active polypeptide with the amino acid sequence shown as SEQ ID NO.7 is derived from Myceliophthora thermophila; the trehalase active polypeptide with the amino acid sequence shown as SEQ ID NO.8 is derived from Rasamsonia emersonii.
The process according to any one of claims 1 to 7, wherein the fermentation product is selected from the group consisting of alcohol and an amino acid selected from the group consisting of glutamic acid, lysine, threonine, valine, proline, tryptophan, isoleucine and leucine, preferably glutamic acid or lysine.
The process according to any one of claims 1 to 7, wherein the trehalose-containing production fluid is selected from the group consisting of a liquefied fluid of an alcoholic fermentation feedstock, an alcoholic fermentation broth, a supernatant of a mature mash of an alcoholic fermentation, a supernatant of an amino acid fermentation or a supernatant of an amino acid fermentation, preferably a liquefied fluid of an alcoholic fermentation feedstock, a supernatant of a mature mash of an alcoholic fermentation, a supernatant of an amino acid fermentation.
The method of claim 1, the step of producing fermentation when the fermentation product is alcohol comprising:

(a) adding amylase to liquefy the alcohol fermentation raw material;

optionally pre-saccharifying the liquefied material prior to step (b);

(b) saccharifying the liquefied feedstock;

(c) adding yeast for fermentation;

(d) after the fermentation is finished, harvesting the mature alcohol mash;

wherein the trehalase may be present and/or added in the following steps:

a saccharification step (b);

a fermentation step (c);

simultaneous saccharification and fermentation;

after fermentation, alcohol is added into the mature mash;

optionally a pre-saccharification step prior to step (b).
The process according to claim 10, wherein the trehalase is added in an amount of 0.05-10U/g DS, preferably 0.1-5U/g DS, more preferably 0.2-0.5U/g DS.
The method of claim 10, wherein step (b) comprises adding a saccharifying enzyme, preferably a complex saccharifying enzyme; said step (c) comprises adding a nitrogen source.
The method according to claim 12, wherein the amylase in the step (a) is high-temperature amylase, and the addition amount is 1-200U/g DS; the saccharifying enzyme in the step (b) is a compound saccharifying enzyme, and the adding amount is 20-600U/g DS; the yeast in the step (c) is active dry yeast, and the adding amount is 100-1500 ppm; the nitrogen source is urea, and the adding amount is 100-1000 ppm.
The method according to claim 13, wherein in the step (a), 10-100U/g DS high-temperature amylase is added to liquefy the alcoholic fermentation raw material; the steps (b) and (c) are synchronously carried out, the pH acidity of the raw material liquefied liquid is adjusted, 50-500U/g DS composite saccharifying enzyme, 200-1000ppm active dry yeast, 600ppm urea and 0.2-0.5U/g DS trehalase are added, and fermentation is carried out for 48-96h at the temperature of 28-36 ℃; harvesting the alcohol matured mash.
The method according to claim 13, wherein in the step (a), 10-100U/g DS high-temperature amylase is added to liquefy the alcoholic fermentation raw material; the steps (b) and (c) are synchronously carried out, the pH acidity of the raw material liquefaction liquid is adjusted, 50-500U/g DS composite saccharifying enzyme, 200-1000ppm active dry yeast and 600ppm urea are added, and fermentation is carried out for 48-96h at the temperature of 28-36 ℃; and (d) harvesting the alcohol matured mash, and adding 0.2-0.5U/g DS trehalase into the supernatant.
The method of claim 1, wherein the step of producing a fermentation when the fermentation product is an amino acid comprises:

(a) seed liquid culture of the fermentation microorganisms;

(b) fermenting and culturing;

(c) collecting fermentation liquor;

wherein the trehalase may be present and/or added in the following steps:

a fermentation culture step (b);

and (c) collecting fermentation liquor.
The method according to claim 16, wherein the trehalase is added in an amount of 0.05-5U/ml, preferably 0.1-2U/ml, more preferably 0.3-1U/ml, most preferably 0.5U/ml.
The method according to claim 16 or 17, wherein the step (a) of shake flask cultivation of the amino acid fermenting strain obtains a seed culture solution; preparing an amino acid fermentation formula in the step (b), sterilizing a fermentation culture medium, inoculating a seed culture solution, and performing fermentation culture for 24-72 hours; the step (c) obtains a fermentation broth.
The process according to claim 18, wherein trehalase is added in step (b) at the start of or during fermentation in an amount of 0.1-2.0U/ml, more preferably 0.2-1.0U/ml, most preferably 0.5U/ml.
The process according to claim 18, wherein trehalase is added to the fermentation supernatant obtained in step (c) in an amount of 0.1-2U/ml, more preferably 0.2-1U/ml, most preferably 0.5U/ml.
The process according to any one of claims 16 to 20, wherein the fermentation product amino acid is selected from the group consisting of glutamic acid, lysine, threonine, valine, proline, tryptophan, isoleucine or leucine, preferably glutamic acid and lysine.
The method of any one of claims 1-7 or 16-20, wherein the fermentation product is an amino acid, and the producing step comprises: adding 0.3-1U/ml trehalase into the fermentation supernatant after the amino acid fermentation is finished, and reacting for 2-7h at pH 6.0-9.0 and temperature 32-39 deg.C.
The method of any one of claims 1-6 or 16-20, wherein the fermentation product is a glutamic acid or lysine, and the producing step comprises: adding trehalase 0.5U/ml into the fermented supernatant after fermentation of glutamic acid or lysine, and reacting at pH 6.5-8.5 and temperature 32-37 deg.C for 5 h.
A method of producing fermented alcohol, the fermenting step comprising:

(a) adding amylase to liquefy the alcohol fermentation raw material;

optionally pre-saccharifying the liquefied material prior to step (b);

(b) saccharifying the liquefied feedstock;

(c) adding yeast for fermentation;

(d) after the fermentation is finished, harvesting the mature alcohol mash;

wherein the method comprises the presence and/or addition of trehalase in the following steps:

a saccharification step (b);

a fermentation step (c);

simultaneous saccharification and fermentation;

harvesting mature alcohol mash after fermentation;

optionally a pre-saccharification step prior to step (b);

the trehalase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
The method of claim 24, wherein the trehalase has the amino acid sequence shown in SEQ ID No.6, SEQ ID No.7, or SEQ ID No. 8.
The method of claim 24 or 25, wherein step (b) comprises adding a saccharifying enzyme, preferably a complex saccharifying enzyme; said step (c) comprises adding a nitrogen source.
The method according to claim 26, wherein the amylase in the step (a) is high temperature amylase, and the addition amount is 1-200U/g DS; the saccharifying enzyme in the step (b) is a compound saccharifying enzyme, and the adding amount is 20-600U/g DS; the yeast in the step (c) is active dry yeast, and the adding amount is 100-1500 ppm; the nitrogen source is urea, and the adding amount is 100-1000 ppm.